The study of the regulation of methionine adenosyltransferase (MAT) and S-adenosylmethionine (AdoMet) in mammalian cells will be studied through the isolation of mutants of Chinese hamster ovary (CHO) cells affected in the synthesis and activity of MAT. The primary objective of the proposed research is the isolation and characterization of MAT-deficient mutants in which MAT activity is absent, reduced or temperature sensitive and regulatory MAT mutants which overproduce MAT and/or AdoMet. Standard mutagenesis techniques will be used to generate desired mutants, which will then be selected using negative methods such as 3H suicide and BUdR killing and positive selection methods relying on increased resistence to toxic compounds involved in AdoMet metabolism. Putative mutants will be screened for growth in selective and non-selective media and further characterized by measurement of both MAT activity and intracellular pools of the compounds involved in AdoMet metabolism. The regulation of MAT activity will be studied in the mutant cells with regard to the effects of exogenous methionine and endogenously synthesized methionine on MAT activity. The existence of different molecular forms or isozymes of MAT will be studied in mutant and parental cells using DEAE-cellulose chromatography and DMSO and AdoMet stimulation of MAT activity to facilitate the detection of changes in the relative amounts of MAT forms in various mutants. Complementation analysis of MAT mutants will also be performed using techniques of somatic cell genetics: cell fusion and detection of specific hybrids by the presence of selectable markers. To isolate CHO mutants having increased competence for DNA uptake, experiments will be initiated with mutagenized TK- cells, using clones TK DNA, and suspected mutants will be further tested with a dominant marker such as methotrexate resistance. Time and progress permitting, the initiation of purification of MAT and the MAT DNA from CHO cells will be undertaken using MAT overproducers and MAT-deficient mutants, including those having increased competence for DNA uptake and expression.